Rabbit retinal organ culture as an in-vitro model of hepatic retinopathy

Abstract

Background: In patients with liver insufficiency the blood ammonia concentration is elevated, which induces structural and functional retinal alterations referred to as "hepatic retinopathy". The fact that some of these alterations are reversible after timely liver transplantation rationalizes therapeutic/preventive approaches to maintain the retina before a donor organ is available. Here, we describe further elaboration and characterization of our retinal organ culture model.

Methods: Retinal pieces of neonatal rabbits were explanted and cultured for up to 22 days. After 7 days in vitro (DIV7), some of the cultures were exposed to 0.25, 0.5, 1.0, 3.0, or 7.0 mM ammonia for varying periods of time and then studied by light and electron microscopy.

Results: Untreated (control) cultures underwent several changes during prolonged culturing; these included increased expression of GFAP and of Bcl-2, and decreased levels of glutamine synthetase, in Müller (glial) cells. However, the retinal layering remained intact and even some retinal ganglion cells survived although their axons had been cut. Exposure to elevated levels of ammonia caused morphologic alterations in the explants compatible with those noted in hepatic retinopathy in vivo. Specifically, we observed neuronal cellular degeneration with destruction of retinal layers and formation of rosettes, as well as decreased expression of intermediate filament proteins, migration of cell nuclei, cell swelling, and up-regulation of glutamine synthetase immunoreactivity in (some) Müller cells.

Conclusion: Our findings indicate that the organ culture model may be a useful tool for detailed studies on the cellular and subcellular mechanisms underlying hepatic retinopathy and for in vitro testing of measures to alleviate HR symptoms.